Our previous studies have indicated that the extracellular matrix protein, vitronectin, directs the localization of both plasminogen activator inhibitor, type I, (PAI-1), and urokinase-type plasminogen activator (uPA) to vitronectin dependent focal adhesion sites on human fibroblasts and sarcoma (HT-1080) cells. The hypothesis to be tested in this proposal is that matrix bound vitronectin will promote plasminogen- dependent cell mediated focal proteolysis of the matrix and subsequent directed cell migration through the matrix. Vitronectin conformers will be compared for their ability to mediate cell adhesion and PAI-1 binding. To determine whether collagen is the binding site for vitronectin in the matrix, the effect of collagenase digestion of fibroblast monolayers on the binding of vitronectin to and the release of vitronectin from the matrix will be tested. The collagen/matrix binding site in vitronectin will be identified by limited protein digestion and deletional analysis of vitronectin cDNA expressed by COS-1 cells. The integrin receptor for vitronectin on HT-1080 cells which triggers uPA receptor clustering will be identified by immunoprecipitation and immunolocalization using vitronectin receptor (alphavbeta3 and alphavbeta5) antibodies. The possible physical linkage of the uPA receptor and the vitronectin receptor of HT-1080 cells will be examined by chemical crosslinking and receptor immunoprecipitations using anti-vitronectin receptor and anti- uPA receptor antibodies. to address the role of vitronectin in focal proteolysis and directed cell migration, a series of mutant vitronectin cDNAs, [normal (wild type) vitronectin (VNwt), vitronectin which cannot bind collagen (VN-coll), vitronectin which cannot bind PAI-1, (VN-PAI-1), or vitronectin which cannot mediate cell adhesion (VN-RGD)] will be constructed by oligo-nucleotide-directed mutagenesis. Vitronectin cDNAs will be transfected into COS-1 cells by DEAE dextran precipitation. All expressed vitronectin proteins will be assessed for the ability to bind collagen and PAI-1, support cell adhesion and direct the clustering of urokinase receptors on HT-1080 cell surfaces. cDNAs directing the synthesis of vitronectins deficient in only one of these activities will be used to transfect HT-1080 cells. Plasminogen dependent proteolysis of each cell line will be measured by plating cells onto radiolabeled matrix molecules and quantifying the release of radioactivity. To determine whether the various vitronectins can localize proteolytic activity to focal areas, cell lines will be plated onto rhodamine conjugated matrix molecules which have been covalently coupled to glass coverslips. If necessary, the conformer of vitronectin required for cell binding will be induced by the addition of thrombin/anti-thrombin III complexes to the assay. Directed migration of transfected cell lines will be assessed using modified Boyden chambers with filters precoated with matrigel or Type I collagen gels. this project will define the potentially important role for vitronectin in the regulation of cell surface proteolysis and will investigate the consequences of this regulation for directed cell migration. These studies will contribute towards the understanding of processes such as angiogenesis and tumor invasion.